Compositions and methods targeting the nucleotide free state of RAS to block oncogenic signaling and transformation

This application is the U.S. national phase application filed under 35 U.S.C. § 371 claiming benefit to International Patent Application No. PCT/US2020/038363, filed on Jun. 18, 2020, which is entitled to priority to U.S. Provisional Patent Application No. 62/862,924, filed Jun. 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

This invention was made with Government support under Grant Nos. R21CA201717-0251, and RO1CA212608 awarded by the National Institutes of Health, and by 2101BX002095 awarded by the U.S. Department of Veteran Affairs. The Government has certain rights in this invention

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Nucleotide-free RAS has a role in regulating specific signaling proteins (Wong et al., 2012, PlosOne, PLoS One. 2012; 7(9): e45360). The ability to inhibit RAS signaling is clinically very important given that Ras proteins are essential components of signaling networks controlling cellular proliferation, differentiation, and survival. Further, oncogenic mutations of the H-ras, N-ras, or K-ras genes are frequently found in human tumors and are known to be oncogenic. Conventional wisdom in the field regarded nucleotide-free RAS as a highly transient state of the RAS protein. Monobodies are single-domain synthetic protein scaffolds that achieve affinity and selectivity similar to antibodies but are refractory to the reducing environment of cells and thus can be utilized as genetically encoded reagents (Koide et al., 2012, J Mol Biol, 415:393-405; Spencer-Smith et al., 2017, Nat Chem Biol, 13(1):62-8; Wojcik et al., 2010, Nat Struct Mol Biol, 17(4):519-27).

Accordingly, there exists a need for improved methods and compositions that that prevent RAS loading with GTP by binding nucleotide-free RAS, for the detection, diagnosis, prevention and treatment of diseases or disorders, including cancer. The present invention meets this need.

In one embodiment, the invention relates to a composition comprising at least one molecule that specifically binds Ras in a nucleotide free state (apo RAS). In one embodiment, the apo RAS binding molecule is a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, an short hairpin RNA, an antisense nucleic acid molecule, siRNA, miRNA, a nucleic acid encoding an antisense nucleic acid molecule, or a nucleic acid sequence encoding a protein.

In one embodiment, the molecule that specifically binds apo RAS a monobody or a fusion protein comprising a monobody domain. In one embodiment, the monobody or monobody domain comprises a peptide sequence of SEQ ID NO: 1-32, a variant thereof, or a fragment thereof. In one embodiment, the fusion protein comprising a monobody domain further comprises a therapeutic agent or a detection moiety.

In one embodiment, the invention relates to a method of treating or preventing a disease or disorder associated with increased levels of apo RAS in a subject, the method comprising the step of administering to the subject a composition comprising at least one molecule that specifically binds Ras in a nucleotide free state (apo RAS). In one embodiment, the apo RAS binding molecule is a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, an short hairpin RNA, an antisense nucleic acid molecule, siRNA, miRNA, a nucleic acid encoding an antisense nucleic acid molecule, or a nucleic acid sequence encoding a protein. In one embodiment, the molecule that specifically binds apo RAS a monobody or a fusion protein comprising a monobody domain. In one embodiment, the monobody or monobody domain comprises a peptide sequence of SEQ ID NO: 1-32, a variant thereof, or a fragment thereof. In one embodiment, the fusion protein comprising a monobody domain further comprises a therapeutic agent or a detection moiety.

In one embodiment, the composition is administered to the subject in combination with a second therapeutic agent.

In one embodiment, the disease or disorder associated with increased levels of apo RAS is cancer.

In one embodiment, the invention relates to an isolated nucleic acid molecule encoding at least one molecule that specifically binds Ras in a nucleotide free state (apo RAS). In one embodiment, the apo RAS binding molecule is a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, an short hairpin RNA, an antisense nucleic acid molecule, siRNA, miRNA, a nucleic acid encoding an antisense nucleic acid molecule, or a nucleic acid sequence encoding a protein.

In one embodiment, the isolated nucleic acid molecule encodes a monobody or a fusion protein comprising a monobody domain. In one embodiment, the monobody or monobody domain comprises a peptide sequence of SEQ ID NO: 1-32, a variant thereof, or a fragment thereof.

In one embodiment, the invention relates to an expression vector comprising an isolated nucleic acid molecule encoding at least one molecule that specifically binds Ras in a nucleotide free state (apo RAS). In one embodiment, the apo RAS binding molecule is a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, an short hairpin RNA, an antisense nucleic acid molecule, siRNA, miRNA, a nucleic acid encoding an antisense nucleic acid molecule, or a nucleic acid sequence encoding a protein.

In one embodiment, the isolated nucleic acid molecule encodes a monobody or a fusion protein comprising a monobody domain. In one embodiment, the monobody or monobody domain comprises a peptide sequence of SEQ ID NO: 1-32, a variant thereof, or a fragment thereof.

In one embodiment, the invention relates to a host cell comprising an isolated nucleic acid molecule encoding at least one molecule that specifically binds Ras in a nucleotide free state (apo RAS). In one embodiment, the apo RAS binding molecule is a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, an short hairpin RNA, an antisense nucleic acid molecule, siRNA, miRNA, a nucleic acid encoding an antisense nucleic acid molecule, or a nucleic acid sequence encoding a protein.

In one embodiment, the isolated nucleic acid molecule encodes a monobody or a fusion protein comprising a monobody domain. In one embodiment, the monobody or monobody domain comprises a peptide sequence of SEQ ID NO: 1-32, a variant thereof, or a fragment thereof.

In one embodiment, the invention relates to a method of diagnosing a disease or disorder in a subject in need thereof, the method comprising: determining the level of apo RAS in a biological sample of the subject, comparing the level of apo RAS in the biological sample of the subject with a comparator control, and diagnosing the subject with a disease or disorder when the level of apo RAS in the biological sample of subject is elevated when compared with the level of apo RAS of the comparator control. In one embodiment, the method further comprises administering a treatment to the subject that was diagnosed as having a disease or disorder.

In one embodiment, the level of apo RAS in the biological sample is determined by contacting the sample with a composition comprising at least one molecule that specifically binds Ras in a nucleotide free state (apo RAS), selected from the group consisting of a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, an short hairpin RNA, an antisense nucleic acid molecule, siRNA, miRNA, a nucleic acid encoding an antisense nucleic acid molecule, a nucleic acid sequence encoding a protein.

In one embodiment, the molecule that specifically binds apo RAS is a monobody or a fusion protein comprising a monobody domain. In one embodiment, the monobody or monobody domain comprises a peptide sequence of SEQ ID NO: 1-32, a variant thereof, or a fragment thereof.

In one embodiment, the level of apo RAS in the biological sample is determined to be elevated when the level of apo RAS is increased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 200%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, or by at least 1000%, when compared with a comparator control.

In one embodiment, the comparator control is a positive control, a negative control, a historical control, a historical norm, or the level of a reference molecule in the biological sample.

In one embodiment, the disease or disorder is cancer.

In one embodiment, the subject is human.

In one embodiment, the invention relates to a method of identifying a modulator of Ras signaling, the method comprising: contacting a sample comprising a Ras polypeptide with a test compound, contacting the sample with a composition comprising at least one molecule that specifically binds Ras in a nucleotide free state (apo RAS). determining the level of apo RAS, and comparing the level of apo RAS in the sample with a comparator control. In one embodiment, the apo RAS binding molecule is a chemical compound, a protein, a peptide, a peptidomemetic, an antibody, an antibody fragment, an antibody mimetic, a monobody, a fusion protein comprising a monobody domain, an aptamer, a ribozyme, a small molecule chemical compound, an short hairpin RNA, an antisense nucleic acid molecule, siRNA, miRNA, a nucleic acid encoding an antisense nucleic acid molecule, or a nucleic acid sequence encoding a protein.

In one embodiment, the molecule that specifically binds apo RAS a monobody or a fusion protein comprising a monobody domain. In one embodiment, the monobody or monobody domain comprises a peptide sequence of SEQ ID NO: 1-32, a variant thereof, or a fragment thereof. In one embodiment, the fusion protein comprising a monobody domain further comprises a therapeutic agent or a detection moiety.

This invention relates to the detection and measurement of nucleotide-free Ras (apo RAS) using an agent that specifically binds to apo RAS. In various embodiments, the invention is directed to compositions and methods for diagnosing, preventing, or treating a disease or disorder associated with aberrant Ras signaling in an individual by performing an assay that measures apo RAS in a biological sample. In various embodiments, the diseases and disorders diagnosable, preventable and treatable using the compositions and methods of the invention include cancers, RASopathies including, but not limited to, neurofibromatosis type 1, Noonan syndrome, Noonan syndrome with multiple lentigines, capillary malformation-arteriovenous malformation syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, and Legius syndrome, and mental disorders including, but not limited to, Alzheimer's disease, Angelman syndrome, autism, cardio-facio-cutaneous syndrome, Coffin-Lowry syndrome, Costello syndrome, Cowden and Bannayan-Riley-Ruvalcaba syndromes, fragile X syndrome, neurofibromatosis type 1, Noonan syndrome, schizophrenia, tuberous sclerosis, and X-linked mental retardation.

In one aspect, the present invention relates to a composition that specifically binds to apo RAS. In one embodiment, the composition comprises an agent that specifically binds to apo RAS, wherein the agent is an apo RAS monobody. In one embodiment, the apo RAS monobody comprises a polypeptide sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32. Therefore, in various embodiments, the invention relates to the detection and measurement of nucleotide free Ras (apo RAS) using an apo RAS monobody. In various embodiments, the invention relates to use of at least one apo RAS monobody of the invention in methods for diagnosing, preventing, or treating a disease or disorder associated with aberrant Ras signaling in an individual. In various embodiments, the diseases and disorders diagnosable, preventable and treatable using at least one apo RAS monobody of the invention include cancers, RASopathies and mental disorders.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well-known and commonly employed in the art.

Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY), which are provided throughout this document.

The nomenclature used herein and the laboratory procedures used in analytical chemistry and organic syntheses described below are those well-known and commonly employed in the art. Standard techniques or modifications thereof are used for chemical syntheses and chemical analyses.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, or ±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected/homeostatic) respective characteristic. Characteristics which are normal or expected for one cell, tissue type, or subject, might be abnormal for a different cell or tissue type.

The term “analog” as used herein generally refers to compounds that are generally structurally similar to the compound of which they are an analog, or “parent” compound. Generally, analogs will retain certain characteristics of the parent compound, e.g., a biological or pharmacological activity. An analog may lack other, less desirable characteristics, e.g., antigenicity, proteolytic instability, toxicity, and the like. An analog includes compounds in which a particular biological activity of the parent is reduced, while one or more distinct biological activities of the parent are unaffected in the “analog.” As applied to polypeptides, the term “analog” may have varying ranges of amino acid sequence identity to the parent compound, for example at least about 70%, more preferably at least about 80%-85% or about 86%-89%, and still more preferably at least about 90%, about 92%, about 94%, about 96%, about 98% or about 99% of the amino acids in a given amino acid sequence the parent or a selected portion or domain of the parent. As applied to polypeptides, the term “analog” generally refers to polypeptides which are comprised of a segment of about at least 3 amino acids that has substantial identity to at least a portion of a binding domain fusion protein. Analogs typically are at least 5 amino acids long, at least 20 amino acids long or longer, at least 50 amino acids long or longer, at least 100 amino acids long or longer, at least 150 amino acids long or longer, at least 200 amino acids long or longer, and more typically at least 250 amino acids long or longer. Some analogs may lack substantial biological activity but may still be employed for various uses, such as for raising antibodies to predetermined epitopes, as an immunological reagent to detect and/or purify reactive antibodies by affinity chromatography, or as a competitive or noncompetitive agonist, antagonist, or partial agonist of a binding domain fusion protein function.

The term “antibody,” as used herein, refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope of a binding partner molecule. Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab, Fab′, F(ab)2 and F(ab′)2, as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to at least one portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, sdAb (either VL or VH), camelid VHH domains, scFv antibodies, and multi-specific antibodies formed from antibody fragments. The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

A “chimeric antibody” refers to a type of engineered antibody which contains a naturally-occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al., Bio/Technology, 9:421). A suitable human acceptor antibody may be one selected from a conventional database, e.g., the KABAT database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. The prior art describes several ways of producing such humanized antibodies (see for example EP-A-0239400 and EP-A-054951).

The term “monobody” as used herein refers to an antibody mimetic or synthetic binding proteins that are constructed using a fibronectin type III domain (FN3) as a molecular scaffold.

“CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy and all light chain CDRs, if appropriate). The structure and protein folding of the antibody may mean that other residues are considered part of the binding region and would be understood to be so by a skilled person. See for example Chothia et al., (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, p 877-883.

The term “framework” or “framework sequence” refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence may be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1, FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. An FR represents one of the four sub-regions, and FRs represents two or more of the four sub-regions constituting a framework region.

As used herein, an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.

By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific binding partner molecule, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to a binding partner molecule from one species may also bind to that binding partner molecule from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to binding partner molecule may also bind to different allelic forms of the binding partner molecule. However, such cross reactivity does not itself alter the classification of an antibody as specific.

In some instances, the terms “specific binding” or “specifically binding”, can be used in reference to the interaction of an antibody, a protein, or a peptide with a second binding partner molecule, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the binding partner molecule; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. In some instances, the terms “specific binding” and “specifically binding” refers to selective binding, wherein the antibody recognizes a sequence or conformational epitope important for the enhanced affinity of binding to the binding partner molecule.

The term “epitope” has its ordinary meaning of a site on binding partner molecule recognized by an antibody or a binding portion thereof or other binding molecule, such as, for example, a monobody. Epitopes may be molecules or segments of amino acids, including segments that represent a small portion of a whole protein or polypeptide. Epitopes may be conformational (i.e., discontinuous). That is, they may be formed from amino acids encoded by noncontiguous parts of a primary sequence that have been juxtaposed by protein folding.

The phrase “biological sample” as used herein, is intended to include any sample comprising a cell, a tissue, or a bodily fluid in which expression of a nucleic acid or polypeptide can be detected. Examples of such biological samples include but are not limited to blood, lymph, bone marrow, biopsies and smears. Samples that are liquid in nature are referred to herein as “bodily fluids.” Biological samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to obtain bodily fluids. Methods for collecting various body samples are well known in the art.

The term “cancer” as used herein is defined as disease characterized by the abnormal growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer (e.g., melanoma), pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, sarcoma and the like.